1. Field of the Invention
The present invention relates to an anti-human lymphotoxin monoclonal antibody, hybridoma cell lines producing said antibody, a process for purification of human lymphotoxin using said antibody, and an immunological assay method and kit using the antibody.
2. Description of the Related Art
Conventionally, antibodies to a particular antigen are prepared by immunizing an animal with the antigen, obtaining blood from the immunized animal and separating an antiserum from the obtained blood. But since an antiserum comprises a plurality of antibodies with different antigen-specificities, it is very difficult to isolate antibodies with a desired antigen specificity.
Further, the specific antibodies thus obtained react with different antigen-determinants and exhibit heterogeneous affinities to the antigen. In 1975, Kohler and Milstein, Nature, 256, 495-497, disclosed a method of obtaining a hybrid cell (hybridoma) which produces an antigen-specific antibody by a fusion between a mouse myeloma cell and an immunized mouse spleen cell, and since that time a large number of hybridomas producing monoclonal antibodies specific to different antigens have been disclosed. These hybridomas are characterized by producing a monoclonal antibody having a predetermined specificity, and can be cloned and stably cultured, and accordingly, there is no limit to the ability to produce an antibody specific to a single antigen determinant.
In a general procedure for constructing a hybridoma, a material containing an antigen to which an intended monoclonal antibody is directed is repeatedly administered to a mammal such as mouse or rat, and after the presence of the intended antibody in serum taken from the animal is confirmed, the spleen is removed from the animal to prepare a cell suspension. Next, the thus obtained cells are placed in contact with myeloma cells, in the presence of a cell fusion promoter such as polyethylene glycol, to fuse the cells. The thus obtained fused cells are then carefully subjected to repeated screening procedures, to isolate a cell line secreting a monoclonal antibody having a desired specificity.
Lymphotoxin is a lymphokine produced by lymphocytes or established lymphoid cells stimulated with an antigen or mitogen, and is promising as an anticancer drug (Granger, G. A. et al., 14th International Congress of Chemotherapy, Kyoto, Japan, Jun. 23-28, 1985, Abstract p15; E. W. B. Jeffes III et al., Lymphokine Research 6, 141, 1987) due to its cytotoxic property to cancer cells; as an antiviral drug (G. H. W. Wong and D. V. Goeddel, Nature, 323, 819, 1986) due to its toxic property to viruses; and as a macrophage activating drug (Japanese Patent Application No. 62-199144), in the medical field. Moreover, other biological activities such as B lymphocyte growth promoting action (J. K. Kehrl et al, Science, 238, 1144, 1987, and an inhibition of lipoprotein lipase activity (Patton, J. S. et al, Proc. Natl. Acad. Sci. U.S.A., 83, 8313, 1986) have been reported. Considering the importance and advantages gained from these biological activities, there is an urgent need to develop accurate and rapid assay methods and assay reagents.
Lymphotoxin can be produced by gene recombination techniques and has been confirmed to have the same lymphotoxin activities as these of a natural lymphotoxin produced by lymphocytes (P. W Gray et al., Nature, 312, 721, 1984; Japanese unexamined Patent Publication, KOKAI No. 62-151182).
The active site of Lymphotoxin is at the carboxyl terminal (c-terminal) side (within 10 amino acids from the c-terminal) in its molecule (Y. Kobayashi et al., J. Biochem., 100, 727, 1986), and various amino terminal-truncated derivatives and variants have been reported (Japanese Unexamined Patent Publication, KOKAI, Nos. 61-181298, 62-258324, 63-5099, 63-8398 and 63-8399). Accordingly, from the viewpoint of control of the process during the production of lymphotoxin, there is also a need to develop accurate and rapid assay methods and assay reagents by which it is possible to separately assay various lymphotoxin variants.
Japanese Unexamined Patent Publications, KOKAI, Nos. 58-16687, 59-84827 and 61-186324 describe methods of purification of natural lymphotoxin obtained by culturing animal cells. Other references, Japanese Unexamined Patent Publications, KOKAI, Nos. 61-56197, 63-3796 and 63-17898, and Japanese Patent Application No. 63-35000 as well as P. W. Gray et al, Nature, 312, 721, 1984, and Bringman, Hybridoma, 6, No. 5, 489, 1987, also disclose purification methods for lymphotoxin.
Among the above-mentioned methods, methods of purification by immuno affinity chromatography using an antibody are described in Japanese Unexamined Patent Publication, KOKAI, No. 61-56197, and reports by P. W. Gray and by Bringman respectively. All of these methods have been created by the same inventors, and use antibodies having the same origin.
Since lymphotoxin exhibits remarkably strong and various physiological actions, there is a desire to obtain specific antibodies directed to an identified recognition site in a lymphotoxin molecule, as a means for the separation, purification and quantitation of lymphotoxin, but lymphotoxins from different animal species have a high homology (Chang - Ben Li, J.Immunol., 138, No. 12, 4496-4501, 1987), and low antigenicity. In particular, the homology of amino acid sequences in the carboxy-terminal side of lymphotoxins, which is essential to lymphotoxin activities, is very high, and therefore, it is difficult to obtain an antibody which recognizes the carboxy-terminal side of lymphotoxin, by using a crude antigen and a conventional immunization method.
Monoclonal antibodies to lymphotoxin are disclosed in Japanese Unexamined Patent Publication, KOKAI, No. 61-56197, and T. S. Bringman, Hybridoma, 6, No. 5, 489-507, 1987, but the antibody obtained in Japanese Unexamined Patent Publication No. 61-56197 is not satisfactorily characterized, and a recognition site in the lymphotoxin molecule for the antibody is not described. According to the report by Bringman, among 13 hybridoma clones, 7 clones secreted antibodies recognizing an amino acid sequence from 7th amino acid THR to 19th amino acid LYS in the amino terminal side of a lymphotoxin molecule shown in FIG. 2. Further Bringman disclosed that antibodies secreted by the above-mentioned hybridoma clones did not react with a polypeptide lacking an amino acid sequence from the first amino acid LEU to the 23th amino acid ALA at the amino terminal of the lymphotoxin molecule.
As mentioned above, it is known that the active site of lymphotoxin is in the carboxy terminal side of the molecule, and various amino terminal truncated derivatives and variants have been reported. The shortest sequences believed to exhibit lymphotoxin activities are those having an amino acid sequence wherein from the first amino acid LEU to the 89th amino acid LYS, or from the first amino acid LEU to the 91th amino acid THR, is deleted (Japanese Unexamined Patent Publication No. 62-258324). It is evident that these amino terminal truncated derivatives and variants of lymphotoxin do not react with the monoclonal antibodies reported by Bringman.
In addition, among 13 clones reported by Bringman et al., although antibodies produced by 6 clones other than the above-mentioned 7 clones were confirmed to be reactive with an amino terminal truncated derivative lacking 23 amino acids at the amino terminal of lymphotoxin, it is not known whether these antibodies react with amino terminal truncated derivatives lacking more than 23 amino acids.
As obvious from the above description, only a limited region in a lymphotoxin molecule can be recognized by antibodies to the lymphotoxin, and a hybridoma cell line which secretes a monoclonal antibody which is reactive with a region other than the above-mentioned amino terminal region of physiologically active lymphotoxin variant, is not known.
The methods of purification of natural lymphotoxin mainly use the properties of glycoside chains attached to the lymphotoxin molecule, but since a recombinant lymphotoxin produced by Escherichia coli has no glycoside chains, the recombinant lymphotoxin cannot be purified by a method applicable to the purification of natural lymphotoxin.
The methods described in Japanese Unexamined Patent Publication Nos. 63-3796 and 63-17898 can be used to purify both the natural lymphotoxin and the recombinant lymphotoxin, but these methods cannot provide a highly purified lymphotoxin.
Although a process described in Japanese Patent Application No. 63-35000 can provide pure lymphotoxin, the process comprises a large number of purification steps, and the operation thereof is complicated and cumbersome and produces only a low yield. Further, the production of a large amount of lymphotoxin requires a large purification plant, resulting in high production costs.
The above-mentioned difficulties accompanying the purification and separation of lymphotoxin are resolved by an immunoaffinity chromatography using an antibody specific to lymphotoxin. Nevertheless, although antibodies used in the immunoaffinity chromatography described in Japanese Unexamined Patent Publication No. 61-56197 are specific to human lymphotoxin, a recognition region in the lymphotoxin molecule has not yet been identified. Namely, in affinity chromatography, these antibodies do not reliably bind amino terminal truncated derivatives and variants while maintaining their lymphotoxin activities.
In a popular lymphotoxin assay method, its cytotoxic activity to mouse L Cells is measured (Y. Kobayashi et al, J. Immunol, 122, 791, 1979).
More specifically, first mouse L 929 cells or substrains thereof are coated in wells of a 96 well microtiter plate to a monolayer and maintained, usually for 15 to 20 hours, and then double-diluted test samples, and actinomycin D to a final concentration of 1 tg/ml are added to the wells, and the mixture is incubated at 37.degree. C. for 20 to 24 hours. Next, the medium is discarded and the cells are stained with a 0.5% crystal violet solution in methanol/water 1:4. After a thorough washing, the dye is extracted from the cells with ethanol, and the absorbance of the extract is measured at 550 mm using a microplate reader or the like. One unit of lymphotoxin activity is defined as an amount which kills 50% of the total number of L 929 cells or substrains thereof, and the activity is calculated from the dilution ratio of a sample.
The above-mentioned conventional lymphotoxin assay is disadvantageous in that the assay takes as long as over 20 hours, the reading fluctuates depending on an extent of the growth of L 929 cells, resulting in a nonreproducible result, and since the assay cannot distinguish between lymphotoxin and tumor necrosis factor (TNF or TNF-a) having similar physiological properties, it is difficult to quantitate lymphotoxin in a sample containing both the lymphotoxin and a tumor necrosis factor, such as blood, urine and the like.
As a means for solving the above-mentioned problems, a quantitation of lymphotoxin by an immunological method using a monoclonal antibody to lymphotoxin has been reported (T. S. Bringman and B. B. Aggarwal, Hybridoma, 6, 489, 1987; A. Meager et al., J.Immunol. Meth., 104, 31, 1987). It is appreciated that, by using such a method, the assay time is shortened and an assay of lymphotoxin becomes possible in the presence of the tumor necrosis factor. Nevertheless, as described above, since a monoclonal antibody specific to an amino terminal truncated lymphotoxin having lymphotoxin activities has not been found, even the above-mentioned method cannot reliably detect amino terminal truncated derivatives and variants of lymphotoxin.
Accordingly, an immunological assay method for lymphotoxin and assay kit therefor using monoclonal antibodies which are reactive with various variants of lymphotoxin essentially having lymphotoxin activities and specific to an identified recognition site in the lymphotoxin variant, is required.